It is estimated that 85% of adults in Western countries have endured dental treatment and by the age of 50 the average adult has lost 12 teeth. Methods are being designed to induce dentin repair and regeneration and more recent research efforts are targeted toward tissue engineering teeth for tooth replacement. In order to successfully induce dentin regeneration or create a tooth in vitro a better understanding of the cells involved needs to be acquired. The goal of this project is to create a relevant model to study dentinogenesis in vitro with the hypothesis that by allowing dental pulp stem cells to generate their own 3D structure in vitro we will create a relevant model system for in vitro studies of the odontoblastic microenvironment and dentin matrix assembly. Stem cells reside in dental pulp that can differentiate to odontoblasts for dentin repair. In situ, odontoblasts reside in three dimensional (3D) structures that they secrete and remodel themselves. Therefore, studies performed with dental pulp cells in a monolayer or with artificially derived scaffolding do not mimic the true microenvironment of these cells. Recent reports in the literature have shown the ability for bone marrow stromal cells to synthesize and remodel their own matrix to create cylindrical bone-like construct. These constructs only consist of the bone marrow stromal cells and their newly made autogenous extracellular matrix. This technology will be utilized to direct the dental pulp cells to create similar scaffold less 3D constructs in vitro. The molecular and structural properties of these constructs will be studied and compared to those created by bone marrow stromal cells. The engineered tissues formed by bone marrow stromal cells and dental pulp cells will then be subcutaneously implanted into immuno-compromised mice to study the effects of the in vivo environment and further verify the creation of dentin- like construct. Public Health Relevance: It is estimated that 85% of adults in Western countries have endured dental treatment and by the age of 50 the average adult has lost 12 teeth. Methods are being designed to induce dentin repair and regeneration and more recent research efforts are targeted toward tissue engineering teeth for tooth replacement. In order to successfully induce dentin regeneration or create a tooth in vitro a better understanding of the cells involved needs to be acquired.